Use of continuous/contiguous stacking hybridization as a diagnostic tool

ABSTRACT

A method for detecting disease-associated alleles in patient genetic material is provided whereby a first group of oligonucleotide molecules, synthesized to compliment base sequences of the disease associated alleles is immobilized on a predetermined position on a substrate, and then contacted with patient genetic material to form duplexes. The duplexes are then contacted with a second group of oligonucleotide molecules which are synthesized to extend the predetermined length of the oligonucleotide molecules of the first group, and where each of the oligonucleotide molecules of the second group are tagged and either incorporate universal bases or a mixture of guanine, cytosine, thymine, and adenine, or complementary nucleotide strands that are tagged with a different fluorochrome which radiates light at a predetermined wavelength. The treated substrate is then washed and the light patterns radiating therefrom are compared with predetermined light patterns of various diseases that were prepared on identical substrates. A method is also provided for determining the length of a repeat sequence in DNA or RNA, and also for determining the base sequence of unknown DNA or RNA.

This application is a Divisional of a Continuation-in-Part (Ser. No.08/855,372) filed May 13, 1997, now U.S. Pat. No. 6,090,549 wherein theparent (Ser. No. 08/587,332) filed Jan. 16, 1996, of said CIP has issuedas U.S. Pat. No. 5,908,745.

CONTRACTUAL ORIGIN OF THE INVENTION

The United States Government has rights in this invention pursuant toContract No. W-31-109-ENG-38 between the U.S. Department of Energy andthe University of Chicago representing Argonne National Laboratory.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a method for analyzing DNA sequences and moreparticularly this invention relates to a method for using sequencing byhybridization with oligonucleotides associated with polyacrylamidematrices, including continuous/contiguous stacking hybridizationmethods, to detect disease-associated alleles.

2. Background of the Invention

Present techniques for determining the existence of disease-associatedalleles in patient DNA are complex, inefficient and somewhat timeconsuming. This is due to the fact that technologies applied to mutationlocation stem from complex and other error-prone base sequencingprocedures. For example, one multi-step DNA sequencing approach, theMaxam and Gilbert method, involves first labeling DNA, and thensplitting the DNA with a chemical, designed to alter a specific base, toproduce a set of labeled fragments. The process is repeated by cleavingadditional DNA with other chemicals specific for altering differentbases, to produce additional sets of labeled fragments. The multiplefragment sets then must be run side-by-side in electrophoresis gels todetermine base sequences.

Another sequencing method, the dideoxy procedure, based on Sanger, etal. Proc. Natl. Acad. Sci. USA 74, 5463-7 (1977) first requires thecombination of a chain terminator as a limiting reagent, and then theuse of polymerase to generate various length molecules, said moleculeslater to be compared on a gel. The accompanying lengthy electrophoresisprocedures further detracts from the utility of this method as a fastand efficient diagnostic tool.

A more recently developed sequencing strategy involves sequencing byhybridization on oligonucleotide microchips, or matrices, (SHOM) wherebyDNA is hybridized with a complete set of oligonucleotides, which arefirst immobilized at fixed positions on a glass plate or polyacrylamidegel matrix. There are drawbacks to this technique, however. Forinstance, given that short nucleotide sequences are repeated ratherfrequently in long DNA molecules, the sequencing of lengthy genomestrings is not feasible via SHOM.

Furthermore, the procedures for manufacturing sequencing microchips withthe required, large number of immobilized oligonucleotides is notperfected. For example, if immobilized octamers are utilized todetermine the positions of each of the four bases in genomic DNA, then4⁸ or 65,536 such octamers need to be fabricated and subsequentlyimmobilized on the gel. Also, hybridization with short oligonucleotidesis affected by hairpin structures in DNA.

Yet another disadvantage in using SHOM is its ineffectiveness indiscriminating perfect DNA-oligomer duplexes from mismatched ones,particularly mismatched duplexes at terminal positions. Such terminalmismatches are harder to discriminate than internal mismatches.

In a variation of SHOM, sequencing of DNA strings is facilitated via acontiguous stacking hybridization (CSH) approach, whereby the microchip,comprising a gel embedded with immobilized oligomer such as an octamer(8-mer), is hybridized first with DNA and then with a fluorescentlylabeled oligomer such as a pentamer (5-mer). The resulting, contiguous13 base-long oligomer (the 5-mer in a juxtaposed position to theimmobilized 8-mer) thus formed acts as a probe to the DNA region.

The efficiency of CSH is due to a more stable probe being formed whenthe immobilized oligomer is positioned side by side with the mobilizedoligomer. This extended complimentary probe therefore results in a morestable duplex between the probe and target DNA.

As with SHOM, however, there are drawbacks with CSH. First, in additionto the 65,536 immobilized oligomers already required to produce theimmobilized oligo fraction in the gel matrix (discussed supra), thenumber of mobile oligomers (i.e. mobile pentamers) necessary tocompletely read the subject DNA via CSH is also formidable. When mobilepentamers are used, for example, given the possibility of any one offour bases at any one base position on the pentamer, all variations ofthe pentamer (4⁵=1,024) must be produced and hybridized with the chip.Furthermore, the microchip, containing the duplexed DNA must becontacted with all the 1,024 pentamers in separate hybridizationprocedures (i.e. performing 1,024 additional hybridization rounds) tofully sequence the subject DNA.

Hybridization of filter-immobilized DNA with oligonucleotides insolution also has been suggested for mutation detection. However, thisapproach is too cumbersome for screening all possible base changes insome genes. For example, in the case of β-thalassemia, the number ofchanges exceeds 100.

A need exists in the art to provide an efficient method for diagnosingdisease by detecting multiple mutation sequences in patient DNA. Such amethod must incorporate a minimal number of oligonucleotides and utilizea minimal number of hybridization steps. The method also must be ofsufficient efficiency so as to effectively discriminate perfect duplexesfrom imperfect ones.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method fordetecting multiple DNA base mutations, which are specific for certaindiseases, that overcomes the disadvantages of the prior art.

Yet another object of the present invention is to provide a method tosequence target DNA by hybridizing the DNA first to oligonucleotidemicrochips and then subjecting the resulting DNA-oligo duplex to mobileoligonucleotides. A feature of the invented method is using a minimalnumber of mobile oligonucleotides to extend the sequences of immobilizednucleotides which are complementary to disease-associated alleles. Anadvantage of the invented method is enhanced detection of theDNA-oligonucleotide duplex.

Still another object of the present invention is to provide a procedurefor more accurately detecting the presence of disease-associated DNAmutations. A feature of the invention is the use of universal bases or amixture of all four bases in oligonucleotide probe sequences. Anadvantage of the method is producing a more sensitive method fordiscriminating perfect duplexes from mis-matched duplexes in SHOMprocedures. Another advantage is to increase the efficiency of CSH byreducing the number of mobile oligomers and hybridization rounds.

Another object of the invented method is to provide a procedure,incorporating a minimum number of stacking hybridization steps, that canaccurately determine the existence of disease-associated DNA mutations.A feature of the method is the simultaneous hybridization of patientDNA, first duplexed with immobilized DNA, with mobile oligonucleotideprobes, each of said probes containing a different fluorochrome. Anadvantage of the invented method is to decrease the number ofhybridization steps, thereby expediting the process of mutationdetection.

Yet another object of the present invention is to provide a method forsequencing DNA and RNA molecules with elongated, immobilized probes. Afeature of the invention is the ligating together of probes after theirjuxtaposition to each other on an immobilization substrate. An advantageof the invention is the ability to sequence-test long DNA and RNAmolecules containing repeat regions. An additional advantage is the useof the invented method to simplify the sequencing of similar genes andgenomes.

Still another object of the present invention is to provide a diagnosticmethod for detecting disease. A feature of the invention is the covalentextension of probes to a target DNA or RNA molecule. An advantage of theinvention is the use of the probes as a diagnostic tool to determine theextent of the existence of repeat sequences in the target molecule, theexistence of which is often proportional to severity of disease coded bythe molecules.

In brief, the objects and advantages of the present invention areachieved by a method for detecting disease associated alleles in patientgenetic material comprising immobilizing a first group ofoligonucleotide molecules of a predetermined length on a predeterminedposition on a substrate, said oligonucleotide molecules synthesized tocompliment base sequences of the disease associated alleles; contactingthe genetic material with said first group of oligonucleotides to formduplexes; contacting the duplexes with a second group of oligonucleotidemolecules, said second group of oligonucleotide molecules synthesized toextend the predetermined length of the oligonucleotide molecules of thefirst group, and where each of the oligonucleotide molecules of thesecond group are tagged with a different fluorochrome which radiateslight at a predetermined wavelength; washing the contacted the duplexes;and comparing the light patterns radiating from the predeterminedpositions on the substrate with light patterns of various diseasesprepared on identical substrates.

Also provided is a method for determining the length of a repeat basesequence in a target oligonucleotide molecule comprising immobilizing afirst end of a starter oligonucleotide molecule; contacting said starteroligonucleotide molecule with the target oligonucleotide molecule so asto cause the target oligonucleotide molecule to hybridize with saidstarter oligonucleotide molecule; contacting a labeled oligonucleotideextender molecule to the target oligonucleotide molecule; allowing saidlabeled oligonucleotide extender molecule to hybridize with a region ofthe target oligonucleotide molecule near a second end of said starteroligonucleotide molecule; determining the base sequence of said regionof the target oligonucleotide molecule that is hybridized with saidlabeled extender molecule; replacing said labeled oligonucleotideextender molecule with an unlabeled oligonucleotide extender moleculehaving the same base sequence as said labeled oligonucleotide extendermolecule; ligating said second end of starter oligonucleotide moleculeto said unlabeled oligonucleotide extender molecule so as to create anew starter oligonucleotide molecule hybridized to the targetoligonucleotide molecule; and repeating the above steps until anonrepeating base sequence of the target molecule is detected.

BRIEF DESCRIPTION OF THE DRAWING

The present invention together with the above and other objects andadvantages may best be understood from the following detaileddescription of the embodiment of the invention illustrated in thedrawings, wherein:

FIG. 1 is a schematic diagram of the stacking between subject matterDNA, gel-immobilized oligonucleotide sequences and oligonucleotideextending sequences, in accordance with the present invention;

FIG. 2 is a schematic diagram of the stacking between subject matterDNA, gel-immobilized oligonucleotide sequences and a plurality of mobileoligonucleotide sequences, each mobile sequence containing a differentdye, in accordance with the present invention;

FIG. 3 is a schematic diagram of the stacking between subject matterDNA, gel-immobilized oligonucleotide sequences, and oligonucleotideextending sequences containing universal bases, or a mixture of all fourbases, and different fluorochromes, in accordance with the presentinvention;

FIG. 4 is a diagram of fluorograms of specific hybridization experimentswith specific immobile and mobile oligonucleotides, in accordance withthe present invention;

FIG. 5 is a diagram of fluorograms of specific hybridization experimentsusing a plurality of tags, in accordance with the present invention;

FIG. 6A is a schematic representation of a target molecule containingrepeat sequences subjected to hybridization-ligation processes, inaccordance with features of the present invention;

FIG. 6B depicts the stability of various hybrid products stacked withprobes illustrated in FIG. 6A;

FIG. 6C depicts the hybridization of target DNA with labeled probes, inaccordance with features of the present invention;

FIGS. 7A, 7B and 7C are radioautographs and fluorographs depicting theefficiencies of the invented phosphorylation and ligation method, inaccordance with the invented method; and

FIG. 8. is a schematic depiction of a method for determining the basesequence of an unknown oligo strand using phosphorylation and ligationof oligomer probes, in accordance with the present invention.

DETAILED DESCRIPTION OF THE INVENTION

A method has been developed to detect DNA mutations associated withspecific diseases. The method involves hybridizing patient geneticmaterial, such as DNA or RNA to a plurality of selected nucleotidepolymers having predetermined lengths, said polymers complementary todisease-associated alleles. The existence of mutations corresponding tospecific diseases are determined by comparing the resulting fluorescenceintensity and or patterns with those patterns which are fingerprints forspecific diseases.

A preferred method is the immobilization of each of the oligonucleotidemolecules in specific array locations on a gel to form microchips. Themicrochips are then sequentially hybridized, with the fragment of DNA(for example PCR product or clone) from an establisheddisease-associated, allele-containing genome, and then with mobile-phaseoligonucleotides that are labeled. Each of said mobile oligonucleotidesmay contain different fluorohromes. After reading the fluorescentpattern, the chip is washed and then subjected to a sample derived fromgenomic DNA (i.e. patient provided) and the same fluorescently labeledmobile phase oligonucleotides. The now-contiguous oligomeric complexthus forms a probe to aberrant DNA regions or mutations. Hybridizationpatterns are then compared to determine the existence of mutations.

An adjunct to the above-described contiguous stacking method involvesextending the length of only those immobilized oligonucleotides that areinvolved in hybridization to thereby increase the sequence efficiency ofthe microchip. The inventors have elucidated a protocol for the sitespecific phosphorylation and ligation of gel-immobilizedoligonucleotides. The combined use of contiguous stacking hybridization,phosphorylation and ligation has resulted in an increased reliability ofsequence measurements and the ability to scrutinize longer-length DNA orRNA.

The above-mentioned phosphorylation and ligation technique isparticularly valuable in sequencing long DNA containing internalrepeats, and therefore identifying unique sequences that flank suchrepeats. Measurements of the number of these repeats is an importanttask since changes in the repeat length in some genes can causegenetically predisposed diseases. The method, in combination with DNAand RNA fractionation techniques also developed by the inventors, isalso valuable for sequence comparison of homologous genomic regionswithout intermediate mapping and cloning.

The invented method obviates the need for the fabrication and arrayplacement of large numbers of immobilized oligomers. Instead, theinvented protocol involves the manufacture of microchips that contain aselection of specific synthetic oligomers, having a length of betweenapproximately 6 and 16 bases, that are immobilized on a gel. Instead ofthe 65,536 immobilized octamers needed to detect every base sequence inan 8-base probe, relatively fewer oligomers, from between approximatelya few dozen to a few hundred, that are specific for disease-associatedallele sequences, are required, depending on the number of fingerprintmutations previously noted in the aberrant gene responsible for thedisease. For example, a microchip with two hundred octamers, which aremanufactured to complement a known allele sequence, and which are alsomanufactured to partly overlap each other by three nucleotides, isutilized to cover a one thousand nucleotide-long DNA molecule, byincrements of five. Patient DNA is hybridized with the microchip tolocalize the pentanucleotide region having a changed structure. Then,successive rounds of hybridization with labeled pentamers, correspondingto the mutations, are utilized to identify the mutation.

FIG. 1 illustrates one embodiment of the invented stacking method. Asdepicted therein, an immobilized oligonucleotide of length L ishybridized with a DNA fragment. When additional oligonucleotides oflength l, l′ and l″ are added, the duplexes formed between all of thepentamers and the DNA are stronger together than if taken separately,particularly when there is a contiguous (uninterrupted) stackinginteraction between L, l, l′ and l″.

The inventors have found that the effective CSH interaction lengths of amicrochip with immobilized octamers hybridized with one, two or threepentamers range from between 13 bases and 23 bases. Generally, a chipcontaining immobilized octamers is hybridized with a solution of targetDNA. This is followed by several rounds of successive hybridizationswith fluorescently-labeled pentamers. Thus, after the target DNAhybridizes first to the immobilized octamer, the same DNA will beavailable to form a duplex with one or more of the pentamers in thesuccessive hybridization steps. Hybridization of each oligomer isdetected by the fluorescence emission of the particular fluorochromethat is coupled to any one oligomer probe.

In another embodiment, depicted in FIG. 2, oligonucleotides l, l′ and l″are labelled with different fluorescent dyes. This allows discriminationbetween duplexes having lengths of for example, 8 nucleotides, 13nucleotides, 18 nucleotides or 23 nucleotides, when immobilized oligomerfractions are 8 nucleotides long and the mobile oligomer fractions are 5nucleotides, or multiples of 5 nucleotides long. The use of differentfluorescent markers allows for simultaneous hybridization of differentmobile fractions, thereby reducing the number of hybridization steps.

The invented CSH method is also applied to identify unknown basechanges. In one instance, this can be accomplished if the complete setof all possible 1024 mobile pentamers is available, for example, infragments 1,000 bases long. The first hybridization is conducted withthe 200 overlapping immobilized oligomers, as discussed supra, topin-point the region where DNA changes exist. Then, hybridization withthe 1,024 mobile pentamers is conducted.

Furthermore, fewer than 1,024 hybridization steps are possible with theinvented method. For example, the number of hybridization steps isdecreased by a factor of four, to 256 steps (i.e. 4⁴=256), when mobilepentamers, which vary from each other in just one base position, areused.

When pentamers containing four universal bases and only one base areused, the number of hybridization steps are decreased to 20. Forexample, complementarity of the mobile oligomer components to thehybridized DNA is imparted by their incorporation of universal bases,such as 5-nitroindole, 3-nitropyrrole,inosine, or all four bases (thefour bases being those found in DNA, namely guanine, cytosine, thymine,and adenine). As a result, successive treatment of the microchip withall possible sequences of the mobile fraction (1024 in the case of apentamer) is obviated. As an example, for the detection of T-basedlocalization in the DNA fragment, only five successive rounds ofhybridization need be performed with pentamers of the followingstructure:

(first round) A-N-N-N-N-fluorochrome A;

(second round) N-A-N-N-N-fluorochrome A;

(third round) N-N-A-N-N-fluorochrome A;

(fourth round) N-N-N-A-N-fluorochrome A;and

(fifth round) N-N-N-N-A-fluorochrome A;

where N designates the universal base (i.e., the four bases A,G,T, andC) and wherein each pentamer is labelled with the same chromophore. Withall four bases to be analyzed, only 20 rounds of hybridization, insteadof 1024, need to be performed.

The use of four different labels decreases the number of necessaryhybridization four times more so that only 5 hybridization rounds needto be performed. In this case, at the first round of hybridization, amixture of four probes is used with the following structure:

A-N-N-N-N-fluorochrome A;

T-N-N-N-N-fluorochrome B;

C-N-N-N-N-fluorochrome C; and

G-N-N-N-N-fluorochrome D.

The color of the resulting spot discloses the substituted base.

To detect the next base, hybridization occurs with another mixture of5-mers, as follows:

N-A-N-N-N-fluorochrome A;

N-T-N-N-N-fluorochrome B;

N-C-N-N-N-fluorochrome C; and

N-G-N-N-N-fluorochrome D.

The third probe has an ATCG sequence at the third position, the fourthprobe has an ATCG sequence at the fourth position, and the fifth probehas an ATCG sequence at the fifth position. This scenario is depicted inFIG. 3.

In addition to the use of universal base or four-base approach,different mobile oligonucleotides, among the 1024 possibilities in thecase of a pentamer mobile fraction, can be labeled with differentfluorochromes. In the case of two different labels, the number ofhybridizations will decrease by a factor of two. In the case of fourdifferent labels, the number of required hybridizations will decrease bya factor of four. This use of different labels is illustrated in FIGS. 2and 3 whereby the geometric shapes of a circle, triangle, pentagon andsquare graphically represent different tags or fluorochromes.

The resulting hybridization of genomic DNAs and pentamers to themicrochips are detected using a multi-wave length fluorescencemicroscope coupled to a CCD-camera. Identification of the allelespresent in the patient genomic DNAs are then determined by analysis ofthe hybridization patterns.

Microchip Manufacturing Detail

Suitable immobilization substrates must have high capacity, berelatively rigid and durable, and should be benign viz hybridization.The use of gel-support for oligonucleotide immobilization offers manyadvantages. Oligonucleotides are tethered into the gel volume instead ofbeing attached to the surface, thereby providing for one hundred timesthe capacity for immobilization (about 1 nmole/mm²) than glass.

A matrix of polyacrylamide gel-elements is prepared by firstpolymerizing a 10-30 μm gel-layer on a glass surface. A myriad oftechniques are available, including that disclosed by K. R. Khrapko etal. J. DNA Sequencing and Mapping Vol 1, pp. 375-388, and incorporatedherein by reference.

Strips of gel are then removed in perpendicular directions to yield gelsquares. Each square is isolated from adjacent squares by strips ofnaked glass, said strips wide enough to prevent accidental mixing ofoligomers. The inventors have found that widths of between approximately80 μm and 200 μm provide good results. A scribing machine facilitatesthis removal, but photolithography methods are also applicable for thepreparation of such gel-square elements. A laser method, developed bythe inventors and disclosed in PCT/RU 92/00052, 1992, incorporatedherein by reference, is also suitable.

Gel-element sizes range from approximately 40 μm×40 μm×20 μm for amicro-chip to 1 mm×1 mm×30 μm for macro-chips. Generally, chip sizesranging from 20 μm×20 μm×20 μm to 1 mm×1 mm×30 μm produce good results.In as much as polyacrylamide gels have low fluorescent background, thesensitivity of the measurements (i.e., the ratio of signal tobackground) will increase with miniaturization of the gel-cell sizes,resulting in an increase in density of the DNA-oligonucleotide duplexes.The inventors were able to detect fluorescence down to 10 amol oflabeled target per 100 μm×100 μm.

Oligonucleotide Synthesis Detail

The synthesis of oligonucleotides for immobilization started from3-methyluridine, located at the 3′-end, as described in Krapko, notedsupra. Oligonucleotides for hybridization were labeled with TMR eitherat the 3′-end by terminal transferase, provided by Promega (Madison,Wis.) and fluorescently labeled dUTP. Alternatively the 5′ amino-groupwas labeled with an excess of N-hydroxysuccinimide ester of5-carboxytetramethylrhodamine (Molecular Probe, lnc. Eugene, Oregon) inDMSO with 50 mM sodium borate buffer, pH 9.0 at 60° C. for 30 minutes.The labeled oligonucleotides were further purified by PAGE and recoveredas described in J. L. Mergny et al. Nucleic Acid. Res. 2 2, 920-928.

The synthesis of oligonucleotides containing universal bases is tosimilar.

Oligonucleotides containing the 3-methyluridine at the 3′ end wereeffective couplers through the aldehyde groups formed after oxidation ofthe 3-terminal ribose residues with sodium periodate. Prior to transferto the gel, up to 2 nmol of oligonucleotide solution is initiallyoxidized in 1 mM to 10 mM NalO₄ at room temperature for 10-100 minutes.Then, ethylene glycol was added to a final concentration of 50 mM.Oligonucleotides were lyophilized, dissolved in water, and then used forspotting, or alternatively, stored in the wells, 2 mm in diameter, ofthe teflon microliter plate, where the oxidation was initially carriedout. Attachment occurs between the oxidized 3′ terminal residue of theoligo and the hydrazide groups of partially modified polyacrylamide gel,whereby the gel was first activated by substituting some amide groupsfor hydrazide ones.

The 3-methyluridine is a good anchor in as much as it forms no stablebase pairs with subject DNA.

Oligonucleotide was applied by robot onto the gel-elements in aliquotsof 1 nanoliter (10⁻⁹ liter) or more. The application technique uses athin thermostabilised metal pin with a hydrophobic side surface and ahydrophilic end-face which determines the spotting volume. Pintemperature is kept close to the dew point to avoid evaporation of thewater solution containing the oligonucleotides.

This transfer technique, developed by the applicants, is more fullydisclosed in PCT/RU #94/000180, incorporated herein by reference.

Once the micro-volumes of the solutions of the bioorganic compounds (theoligomers) have been applied to all cells of the matrix, themicro-matrix temperature is set equal to or below the dew point of theambient air. The temperature is maintained until swelling of the gel iscomplete and non-coalescent droplets of water-condensate appear in thespacings between the cells. Then, a thin layer of an inertnon-luminescent oil (such as NUJOR Mineral Oil from Plough, Inc.) iscautiously applied to the micro-matrix surface, the thickness of thelatter layer being over 100 μm. The micro-matrix is kept under the oillayer until the immobilization process is complete, preferably for atleast 48 hours. The oil is then removed with a solvent, such as ethanoland water, and the matrix is dried and stored ready-for-use. Moreelaborate discussion of the foregoing matrix preparation procedure isfound in PCT/RU94/00178, incorporated herein by reference.

The bond between an oligonucleotide and polyacrylamide is stabile enoughfor the matrix to withstand 10-15 rounds of hybridization without anynoticeable degradation. The half-life of the oligonucleotide-gel bond at60° C. is 2 hours, and at 25° C., 36 hours.

Oligonucleotides are immobilized on the gel in spaced arrays so as toprevent interference during hybridization and enhanced hybridizationefficiencies. Gel-loading capacity limits of between 0.01 percent and 30percent yield good results, with a preferable range of betweenapproximately 0.1 percent and 10 percent. Concentrations of the oligo tothe subject DNA can vary, and generally range from between 100 to 1,000times higher in concentration compared to the subject DNA. Convenientsubject DNA concentrations range from 0.1 to 1 picomole (pmole=10⁻¹²moles) in one microliter, and a typical oligo concentration is 100micromoles (fmole=10⁻¹⁵ moles) per gel element of 100 squarecentimeters.

It was observed that more than 70 percent of gel-immobilizedoligonucleotides formed duplexes with DNA. The effective temperaturestability of duplexes formed between DNA and gel-immobilizedoligonucleotides depend on their concentrations and base-pair lengths.Generally, the inventors obtain good DNA complexing with immobilizedoligomers at temperatures ranging from between approximately 0° C. and60° C. Duplexing is further enhanced at high temperatures whenoligonucleotides with relatively long base-pair lengths (e.g. 10-mersand 12-mers) are used. For example, when using immobilized pentamers,good duplexing occurs at between 10° C. and 20° C. When usingimmobilized octamers, preferable temperatures are selected from a rangeof between approximately 25° C. to 45° C. across the 0.01 percent to 30percent gel capacity spectrum. This flexibility in gel loading providesthe ability to equalize the stability of AT- and GC-rich duplexes ininstances where universal bases are not used but where a plurality ofdifferent fluorochromes are utilized.

The inventors have found that the incorporation of additional universalbases in the mobile oligomer fraction stabilizes pentamers. Essentially,the incorporation of said universal bases turns terminal mismatches intointernal mismatches, which are more easily discriminated from perfectduplex images.

Hybridization, Washing and Staining Detail

All procedures were performed on a Peltie thermotable. Hybridization ofa microchip with fluorescently labeled DNA was carried out at 0° C. for30 minutes in 1 μl of washing buffer with 1 percent TWEEN 20(Calbiochem, La Jolla, Calif.) detergent or any other detergent,specifically a detergent containing polyoxyethylenes. Washing buffercontained 1 M NaCl, 5 mM Na phosphate (pH 7.0), 1 mM EDTA. Thereafter100 μl of washing buffer was pipetted on the microchip at 0° C. for 10seconds and carefully pipetted off to remove unhybridized DNA. Thewashing could be repeated by varying the temperature and duration.

Contiguous stacking hybridization is carried out by hybridizing themicrochip with 1 μmole of unlabeled target at 0° C. for 30 minutes, andoptionally, washed at 15° C. for 2 minutes, as described, supra. Thesecond round of hybridization was carried out with 1 μl mixture offluorescently labeled 5-mers (5 pmol each) at 0° C. for 10 minutes. Thematrix was rinsed once with washing buffer with 1 percent TWEEN 20.While a washing step is not usually necessary, any washing proceduresemployed usually encompassed washing off the hybridized 5-mers at 15° C.for 2 minutes, and hybridization with the other mixtures of 5-mers wasrepeated under the same conditions.

Fluorescence Analysis Detail

A multi-wave length fluorescence microscope coupled with a CCD-camerawas assembled for image analysis. An objective yielding a 3-mmobservation field enabled the simultaneous analysis of over 1,000elements of the microchip at once. Specifically, the microscope (350 Whigh pressure mercury lamp, Ploem opaque with interference excitationand barrier filters for TMR) equipped with special optics and a CCDcamera was built. The 3×objective with the 0.4 numerical aperture allowsthe illumination of the object field up to 7 mm in diameter and project2.7×2.7 mm of the microchip on the CCD matrix. The CCD head is similarto that manufactured by Princeton Instruments (Trenton, N.J.). Theexposure time varied from 0.4 to 30 seconds with a readout time of about1.3 seconds. Variation in the sensitivity within the object area did notexceed 5 percent. The system allows operation with 1.7×objective withthe same numerical aperture for analyzing 5×5 microchip areas. Theinstant configuration allows for rapid change-out of Filters fordifferent fluorochromes.

The image of the microchip on the CCD camera was accommodated by amicrocomputer, similar to the configuration disclosed in Khrapko, K. R.et al. J. DNA Sequencing and Mapping, 1, pp. 375-388, and incorporatedherein by reference. For printing, linear transformation was used. Thisbrought the highest pixel values to the same level for all images. Fordigital estimation, the image of the microchip element was fully coveredby a ‘square’ twice the size of the element. Then a frame wasconstructed around the ‘square’ with equal square area. The signal ofthe element was calculated as the signal from the square minus thesignal from the frame.

Fluorochrome Detail

Tetramethylrodamine (TMR) was used as a dye for labeling either the DNAor the mobile oligomers. Other dyes that are suitable labeling compoundsinclude, but are not limited to, fluoresceine, Texas Red, Cascade Blue,and rhodamine, all available from Molecular Probes. HEX™, marketed byApplied Biosystems in Foster City, Calif., is another suitable dye. Inthe case of DNA-labeling, before measurements the microchip is incubatedwith fluorescent tagged DNA at 0° C. for 30 minutes and then rinsed for10 seconds with washing buffer to remove unbound targets. Perfectduplexes are discriminated already in the process of hybridizationdespite rather high intensities of the fluorescence signal from theunbound target. More effective discrimination of perfect duplexes frommismatched ones are achieved by plotting the dissociation curve eitherat temperature gradient or at a fixed temperature while changing theduration of washing. Real-time measurements allowed for the choice ofoptimal conditions for discrimination when the mismatched signals areclose to background levels. Temperatures are controlled by a Peltiethermotable.

PCR Detail

PCR amplifications were performed by an adapted procedure by Postnikovet al. Hemoglobin 1 7,439-453 (1993), and incorporated herein byreference. Initially, amplification of 421 bp-long product was carriedout with 1 ng of genomic DNA, primers #12005 TGCCAGAAGAGCCAAGGACAGG(SEQ. ID. No:81) and #12406 TMGGGTGGGCCCCTAGACC, (SEQ. ID. No:81) Thereaction conditions were as follows: 30 cycles with 40 seconds at 93°C., 30 seconds at 67° C. and 30 seconds at 72° C. 5 μl of the PCR weretransferred to the reaction mixture for amplification with nestedprimers. Nested primers, #12156 ATUTGCTTCTGACACAACT (SEQ. ID. No:83) and#12313 TCTCCTTAAACCTGTCTTG, (SEQ. ID. No:84) were used to amplify 176bp-long DNA for 25 cycles (30 seconds at 90° C., 30 seconds at 50° C.,20 seconds at 72° C.). The PCR with fluorescently nested primers(labeled #12272 CCCTGGGCAG (SEQ. ID. No:85) and normal #12299GTCTTGTAACCTTG) (SEQ. ID. No:86) was carried out for 25 cycles (30seconds at 80° C., 30 seconds at 35° C.) and yielded 32 bp product. PCTwas purified by PAGE or by enrichment procedures.

EXAMPLE 1

Two 8-mers located one and two bases away from a mutation site wereimmobilized on a microchip, and the microchip was hybridized to theunlabeled 19-mer. Then the duplexes formed on the microchip werehybridized in three more rounds with pools of two labeled 5-mers. Theresults are illustrated in FIG. 4. Some hybridized pentamers formedperfect duplexes in a juxtaposed position to the immobilized 8-rner andextended it to a 13 bp-long duplex. These 13-base perfect duplexes werestable due to stacking interactions between 5′- and 3′ terminal bases ofthe 8-iner and 5-iner, respectively, despite lacking a phosphodiesterbond. Mismatches in the internal, or even in the terminal, positiondestabilized the interaction of the 5-mers much more than 8-mers.

As can be noted in FIG. 4, the mismatched 5-mers were either nothybridized at all or washed out at much lower temperature than fullycomplementary 5-mer. Therefore, inclusion of 5-mers provided betterdiscrimination of perfect duplexes from mismatched ones than justimmobilized 8-mers, particularly in the case of terminal mismatches. The8-mer duplexes remained stable under the washing conditions for thepentamers. The microchips sustained up to 10 rounds of successivehybridization with 5-mers. The 5-mers can also be ligated to 8-mersenzymatically. However, the ligation could complicate those experimentswhere several rounds of CSH are to be performed.

EXAMPLE 2

FIG. 5 depicts another embodiment wherein a plurality of tags orfluorochromes are used to detect target sequences. In this embodiment, amicrochip was constructed, consisting of 10 immobilized octamers, withsequences engineered to affect a three-base overlap. For octamersequence strings labeled “1.”, “2.”, and “3.”, the sequences varied byeither a cytosine or thymine, as indicated by the underscore.

The microchip was hybridized with a 21-nucleotide-containing sequence(21-mer). After the hybridization procedure, unhybridized 21-mer wasremoved and the microchip was hybridized with a mixture of HEX-labeledpentamer ACCTT and FAM-labeled pentamer GATAC, at a labellingconcentration of 50 percent of total pentamer.

Detection of the HEX- and FAM-tags (designated as * and Λ, respectively)was performed by changing to the corresponding sets of optical filters.

The octanucleotide labeled as “−1.” was constructed as a negativecontrol, in as much as this string did not interact with a pentamer.Octamer “+1” was constructed as a positive control for hybridization,and for proper filter function.

As can be noted in FIG. 5, the invented contiguous stacking method ofusing a plurality of tags provides excellent detection of targetsequences, and with no false negatives or positives. For example, String“1.”, depicting an immobilized octamer stacked with two mobile, anddifferently-labeled pentamers (in italics), clearly revealed thepresence of the target sequence when each tag was utilized.

Hybridization-Lioation Detail

In one exemplary embodiment, discussed infra, a decamer (10-mer)flanking five repeats from the 3′ end, is first immobilized onto a gelelement. The 10-mer is hybridized with a target ssDNA and thensuccessively ligated with four different unlabeled phosphorylatedpentamers (5-mers). Each step of the ligation is controlled byhybridization with fluorescently labeled 5-mers. Lastly, a resultingmulti-oligomer probe (a 30-mer in the case when four different pentamersare ligated to the already immobilized decamer) is hybridized with a 5′labeled pentamer to establish repeat length.

Alternatively, only one oligonucleotide can be used forhybridization-ligation walking if its length is a multiple of repeatlengths. For example, a hexamer could be employed for dinucleotide andtrinucleotide repeats; a tetramer or octamer can be used fortetranucleotide repeats. Unphosphorylated oligonucleotide and enzymaticphosphorylation are introduced at each walking step to avoidsimultaneous coligation of several copies of the oligonucleotide. Afterthe first step of hybridization with DNA, and the second step ofligation of phosphorylated immobilized oligonucleotide with theunphosphorylated repeat oligomers, the ligated product is phosphorylatedto carry out the next hybridization-ligation step. This process issuitable in measuring the length of DNA containing tens and hundreds ofshort repeats, with the 10-50 base pair long blocks of these repeatsbeing used at each step of the hybridization-ligation “walking”procedure.

EXAMPLE 3

Hybridization-Ligation

ssDNA containing five tetranucleotide 3′-ACAT-5′ repeats was used as atest model. Successive hybridization-ligation “walking” of fivepentamers P1-P5 (5-mers) along this ssDNA, first hybridized to animmobilized decamer (10-mer), was performed to measure the repeatlength.

As a first step, the decamer was immobilized in each of four gelelements. The decamer-loaded gel element was subsequently hybridizedwith unlabeled DNA and then contacted with a first fluorescently labeledpentamer. Stability of the first pentamer is enhanced by stackinginteractions of adjacent bases at the terminal positions of the firstpentamer and the decamer. Other pentamers, which form gaps with thedecamer at this particular point of the hybridization sequence are nothybridized due to the lack if stabilizing stacking interactions.

The first labeled pentamer is then washed off the gel element andreplaced with an identical pentamer lacking the label. This unlabeledpentamer is ligated to the gel-immobilized decamer (previouslyphosphorylated), resulting in a 15-mer polymer probe being formed.

In a second round of “walking”, a second fluorescently labeled pentameris hybridized with the DNA at the 5′ end of the formed 15-mer. Afterhybridization, the second labeled pentamer is removed and replaced witha second unlabeled pentamer. This second unlabeled pentamer is thenligated to the 15-mer polymer to form a 20-mer polymer. Two more roundsof hybridization and ligation “walking” extends the length of themicro-chip immobilized polymer to 25 and 30 base lengths, respectively.

The final hybridization of the microchip containing the 30-merimmobilized polymer is carried out with DNA and a fifth labeled pentamerthat is complementary to the 3′ flanking sequence of the repeat region.This results in the determination that the length of the tetranucleotiderepeat in the target DNA is 20 bases (4×5=20).

FIG. 6 provides a schematic view of the repeat length determiningprocess. FIG. 6A shows the results of hybridization-ligation of ssDNAcontaining five tetranucleotide 3′-ACAT-5′ repeats. The successivehybridization-ligation “walking” of five pentamers (P1=5′-GTATG-3′,P2=5′-TGTAT-3′, P3=5′-ATGTA-3′, P4=5′-TATGT-3′ and P5=5′-AATTG-3′) alongthe ssDNA, first hybridized to a chip-bonded 10-mer, was carried out tomeasure the number of repeats. The decamer (5′-TGATGACTGG-3′) (SEQ. ID.NO.87) complementary to the ss DNA was immobilized in four adjacent gelpads of the chip, as depicted in FIG. 6C.

The chip, as depicted in FIG. 6C(1) was first hybridized with unlabeledDNA and the fluorescently labeled 5-mer P1. As can be noted in FIG. 6B,column P0 (P0 which denotes hybridization but no ligation) P1 and thedecamer formed a stable contiguous 15 basepair duplex with the DNA. P1was stabilized in the duplex by the stacking interactions of adjacentbases at the terminal positions of P1 and the decamer. As can be notedtraveling down the P0 column, the other pentamers (P2, P3, P4, and P5)are not prone to hybridization due to lack of stabilizing stackinginteractions.

All of the labeled pentamers were washed off the chip and the unlabeledpentamer P1 was added and ligated to the decamer to create acomplementary 15-mer immobilized fraction that is complementary to theDNA. To this 15-mer (depicted in column P1) was added labeled pentamerP2, resulting in an AU intensity of 70 for P2. However, due toincomplete ligation in the previous cycle, a fluorescence intensity of51 AU (arbitrary units) for P1 was also observed.

As in the previous step, the labeled P2 was washed from the chip andunlabeled P2 was ligated to the existing 15-mer to form a 20-merimmobilized fraction. When labeled P3 was added to the 20-mer, anintensity of 45 was observed (see column+P2). Two additional cycles ofhybridization and ligation “walking” with P3 and P4 resulted inelongating the 20-mer to a 25-mer and a 30-mer immobilized oligo. Finalhybridization of the 30-mers formed after the four ligations was carriedout with P5 which is not complementary to the repeat sequence in theDNA. Therefore, this final hybridization determines the sequence whichflanks the 3′ end of the repeat region of the DNA while also determiningthe length of the tetranucleotide repeat as 4×5=20 bases.

Site-directed Phosphorylation and Ligation of Immobilized Oligomers

The results of site-directed phosphorylation and ligation on specifiedpads of a gel pad are shown in FIG. 7. T4 polynucleotide kinase (140kDa) and T4 DNA ligase (54 kDa) diffused into the four percentpolyacrylamide gel of the microchip pads to catalyze phosphorylation andligation of immobilized oligonucleotides. A ³²P-labeled decamer wasimmobilized within element “A” and the same nonphosphorylated decamerwas attached to the “B”, “C” and “D” pads of the array depicted in 7A.

The mixture of the kinase and γ-³²P-ATP in a buffer was added only toelement “B”. The same solutions, but lacking kinase, were added to the“C” element to establish a control. After incubation of the array at 37°C. for 3 hours, the ³²p label appeared only in element “B”. That nocrosstalking of element “B” with “C” or “D” was observed confirms thatthe invention provides for efficient and site-directed phosphorylationof gel-immobilized oligonucleotides.

Complementary 21 base-long DNA, fluorescently labeled pentamer and DNAligase were added to the immobilized, phosphorylated decamers (element“B”) and immobilized, nonphosphorylated decamers (element “C”). The samesolution, but not containing ligase, was added to control elements “A”and “D” containing phosphorylated and nonphosphorylated decamers. Aftercompletion of ligation at 4° C. for 5 hours and subsequent washing, thefluorescence label was observed only in element “B”, as depicted in FIG.6c. The results of the ligation experiments substantiated the inventedprotocol providing for immobilized oligonucleotide phosphorylation andligation in predefined areas of an oligonucleotide microchip.

A more detailed description of the phosphorylation and ligationprotocol, including the production of controls is as follows: Fiftypicomoles of synthetic oligodeoxyribonucleotide(5′-ATACCAACCT-r^(3m)U-3′) (SEQ. ID. NO. 88) was phosphorylated within10 microliters (μl) of a reaction mixture containing 1×PNkinase buffer(available from Epicentre Technologies, Madison, Wis.), 15 μCi [γ-³²P]ATP, 500 pmol ATP and 0.5 U T4 Polynucleotide kinase (also fromEpicentre Technologies) at 37° C. for 60 minutes. The ³²P-labeleddecamer was purified from unincorporated label with Bio-Spin 6Chromatography Column (Bio-Rad Laboratories, Hercules, Calif.) andimmobilized within the “A” gel element of FIG. 6a. The same oligomer,but not phosphorylated, was immobilized within the “B”, “C” and “D” padsof the chip depicted in FIG. 6a. Immobilization was carried out at 20°C. for 12 hours. The chip was washed with washing buffer comprising 0.2M NaCl, 0.2 mM EDTA, 2 mM sodium phosphate, pH 6.8 at 37° C. for onehour and then rinsed with water. The chip was dried and radioautographedwith Kodak Scientific Imaging Film X-OMAT.

One microliter of the phosphorylation mixture 1.5 μCi [γ- ³²P] ATP, 50pmol ATP and 0.05 U T4 Polynucleotide kinase in 1×PNkinase buffer wasadded to gel element “B”. As a control, either the same mixture(excluding kinase) was added to pad “C” or 1 μl of 1×PNkinase buffer wasadded to gel cell “A”. Enzymatic phosphorylation was carried out at 37°C. for 3 hours at 100 humidity. The chip was washed first with washingbuffer (0.2. M NaCl, 0.2 mM EDTA, 2 mM sodium phosphate pH 6.8) at 37°C. for 1 hour, then with water, dried and radioautographed. 2.5 μl ofligation mixture containing 10 pmol of ss DNA(5′-TGGGCAGGTTGGTATCAAGGT-3′) complementary to the immobilized decamer,50 picomol of fluorescently labeled pentamer (5′-HEX-CCTTG-3′) stackedto the immobilized decamer, 1 mM ATP, and 0.1 U T4 DNA ligase (fromEpicentre Technologies) in 1×T4 DNA ligase buffer (also from Epicentre)were each added to the “B” and “C” gel pads. As a control, 2.5 μl of thesame mixture, excluding ligase, was added to the “A” and “D” gelelements. Ligation was carried out at 4° C. for 5 hours, then the chipwas washed with washing buffer (0.2 M NaCl, 0.2 mM EDTA, 2 mM sodiumphosphate, pH 6.8) at 10° C. for 5 minutes.

Four pentamers, a decamer and single stranded DNA were synthesized. Alsosynthesized were the same four pentamers and a fifth pentamer, all fiveof which contained fluorescein at their 5′ end.

One picomole of the decamer and 40 picomoles of each of the first fourpentamors were phosphorylated separately with 10-fold excess of ATP and1 U of T4 Polynucleotide kinase. The phosphorylated decamer wasimmobilized within four gel pads (100 fmol per 100×100×20 μm pad) on amicromatrix. Four rounds of successive contiguous stacking hybridization(CSH) followed by ligation were carried out under similar conditions.

The first hybridization was carried out in 10 μl of hybridization buffercontaining 1 μM fluorescently-labelled first pentamer and 1 μM ofcomplementary ssDNA. Approximately 10 ml of the hybridization solutionwas placed on the microchip, and the chip covered with a cover-glassslide over 0.1 mm-thick spacers to be incubated for 5 minutes at 7° C.After the hybridization, the cover glass was removed and the microchipwas washed with distilled water for 10 minutes at 37° C. to remove alllabelled pentamer. Four microliters of ligation mixture containing 4pmole of the ssDNA, 40 pmole of the first phosphorylated pentamer, and 1U T4 DNA ligase in 05.×dilution and 1×ligation buffer (available in theRapid DNA Ligation Kit from Boehringer Mannheim (Indianapolis, Ind.)were placed on the microchip. Ligation was carried out at roomtemperature for 4 hours at 100 percent humidity. After completing thereaction, the chip was washed for 10 minutes at 37° C. with distilledwater and dried. The next three rounds of hybridization-ligation werecarried out with a second pentamer, then a third, and finally a fourthpentamer. Lastly, the ligated 30-mer of the microchip was hybridizedwith DNA and the labeled fifth pentamer.

Sequencing of Long DNA/RNA Molecules

Aside from determining the length of repeat sections of DNA or RNAmolecules, determining the sequence of unknown DNA or RNA molecules alsois a high priority. Sequencing efforts using gelmatrices have beenrelegated to the sequencing of strands of approximately 200 bases inlength. This is due to the fact that each cell in a gel matrix mustcontain only one oligomer. While sequencing sensitivity is proportionalto oligomer length, an increase in oligomer length also requires morecells per gel matrix. For example, the use of immobilized hexamersrequires that 4096 cells (4⁶) be constructed, each to hold just onepossible hexamer sequence. If octamers are used, then 65, 336 (4⁸) cellsare required.

The present method allows a sequence determination to occur atsensitivities provided by a 13-mer (or higher base number) probe, butwithout the concomitant requirement for a large number of cells in a gelmatrix pattern. For example, if in a first step, an unknown targetstrand of DNA or RNA is hybridized on an oligomer chip and two identicaloctamers are found on the strand, a need exists to determine theflanking regions surrounding those oligomers. In this scenario, theregion containing the target strand duplexed with one of the octamerscan be exposed to a pool of labeled pentamers in an effort to extend theoctamer sequence to a 13-mer, an 18-mer or longer. The appropriatecomplementary pentamers, once identified via stacking hybridization, arethen ligated onto the original immobilized probe (after the originalimmobilized probe is phosphorylated) to form a longer probe. Theexistence of this new, extended probe provides the sensitivity of a gelmatrix containing all variants of the now-formed, longeroligonucleotide.

After construction of this longer probe, contiguous stackinghybridization then can be used in conjunction therewith to reconstructlarger fragment sequences of the target molecule.

EXAMPLE 4

Sequencing Unknown ssDNA Using Phosphorylation-Ligation Technique

FIG. 8 illustrates the utility of the probe-extending technique todetermine the base sequence of an unknown target oligonucleotide strand.An exemplary 75-base fragment containing two sequences, each repeatedonce, was sequenced using the method. The 75-base fragment is designatedas number 810 in FIG. 8. The two different sequences 812 and 814 are inbold and underlined, respectively.

The 75-base fragment 810 is subjected to a hybridization chip (notshown) containing all possible octamers. As a result of thehybridization step, a list, 816, of all heptamers (7-mers), withportions of immediately adjacent oligos, in the original 75-basefragment is generated. As can be noted, the sequence designated as “1.”in the list 816 corresponds to the first seven bases of the 75-basefragment. The sequence designated as “2.” in the list corresponds to theseven bases in the 75-base fragment starting with the second basethymine. Correspondingly, the sequence designated as “3.” in the listcorresponds to the seven bases in the 75-base fragment starting with thethird base adenine. That the hybridization list, 816, contains only 46unique heptamers indicates that some subfragments dwelling in the75-base fragment are repeated.

Using the heptamer list 816 discussed above, seven subfragments havingdefinitive base sequences are determined to exist in the 75-basefragment. A list, 818, of the seven subfragments, (i.e. subfragmentnumbers 1-7) further indicate (in the “frequency” column) the number oftimes each fragment appears in the 75-base fragment. Also determinedfrom the subfragment analysis is which subfragments appear before andafter a particular subfragment. A pictorial of this subfragment list andthe relative positioning of subfragments viz. one another are depictedin 820 of FIG. 8.

The “before”, “after”, and “frequency” information of the subfragmentlist is then utilized to construct two possible 75-base sequences, 822and 824, the first sequence 822 of which is the correct sequence. Alsoaccompanying the two possible sequences, are the fragment positions. Ascan be observed, simply employing contiguous stacking hybridizationusing two pentamers with an immobilized octamer to produce an 18-merhybrid will not result in differentiating between the original 75-basesequence 822 and the other 75-base possibility 824. As such, the octamerfirst must be phosphorylated and then ligated with two pentamers toproduce an 18-mer probe, to which contiguous stacking hybridization canbe applied to definitively determine the correct sequence.

In summary, the aforementioned method of phosphorylating and ligatingoligos to form a foundation for later CSH is a powerful method fordefinitively sequencing long DNA or RNA molecules or similar structures.

While the invention has been described with reference to details of theillustrated embodiment, these details are not intended to limit thescope of the invention as defined in the appended claims.

88 19 bases nucleic acid Not Applicable linear Genomic DNA yes 1CCTGGGCAGG TTGGTATCA 19 8 bases nucleic acid Not Applicable linearGenomic DNA yes 2 TGCCCAGG 8 5 bases nucleic acid Not Applicable linearGenomic DNA yes 3 CAACC 5 5 bases nucleic acid Not Applicable linearGenomic DNA yes 4 CAAGC 5 5 bases nucleic acid Not Applicable linearGenomic DNA yes 5 CAACC 5 8 bases nucleic acid Not Applicable linearGenomic DNA yes 6 CTGCCCAG 8 5 bases nucleic acid Not Applicable linearGenomic DNA yes 7 CCAAC 5 5 bases nucleic acid Not Applicable linearGenomic DNA yes 8 CCAAC 5 5 bases nucleic acid Not Applicable linearGenomic DNA yes 9 CCAAA 5 21 bases nucleic acid Not Applicable linearGenomic DNA yes 10 TGGGCAGGTT GGTATCAAGG T 21 8 bases nucleic acid NotApplicable linear Genomic DNA yes 11 AACCTGCC 8 18 bases nucleic acidNot Applicable linear Genomic DNA yes 12 ACCTTGATAC CAACCTGC 18 8 basesnucleic acid Not Applicable linear Genomic DNA yes 13 CCAACCTG 8 8 basesnucleic acid Not Applicable linear Genomic DNA yes 14 ACCAACCT 8 8 basesnucleic acid Not Applicable linear Genomic DNA yes 15 TACCAACC 8 8 basesnucleic acid Not Applicable linear Genomic DNA yes 16 ATACCAAC 8 13bases nucleic acid Not Applicable linear Genomic DNA yes 17 ACCTTGATACCAA 13 40 bases nucleic acid Not Applicable linear Genomic DNA yes 18GTCCCCAGTC ATCACATACA TACATACATA CATACAATTT 40 10 bases nucleic acid NotApplicable linear Genomic DNA yes 19 TGATGACTGG 10 5 bases nucleic acidNot Applicable linear Genomic DNA yes 20 GTATG 5 5 bases nucleic acidNot Applicable linear Genomic DNA yes 21 TGTAT 5 5 bases nucleic acidNot Applicable linear Genomic DNA yes 22 ATGTA 5 5 bases nucleic acidNot Applicable linear Genomic DNA yes 23 TATGT 5 5 bases nucleic acidNot Applicable linear Genomic DNA yes 24 AATTG 5 7 bases nucleic acidNot Applicable linear Genomic DNA yes 25 CTAAGTG 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 26 TAAGTGT 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 27 AAGTGTG 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 28 AGTGTGG 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 29 GTGTGGC 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 30 TGTGGCG 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 31 GTGGCGG 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 32 TGGCGGA 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 33 GGCGGAA 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 34 GCGGAAC 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 35 CGGAACT 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 36 GGAACTA 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 37 GAACTAC 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 38 AACTACG 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 39 ACTACGT 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 40 CTACGTC 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 41 TACGTCC 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 42 ACGTCCT 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 43 CGTCCTC 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 44 GTCCTCT 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 45 TCCTCTA 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 46 CCTCTAA 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 47 CTCTAAC 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 48 TCTAACA 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 49 CTAACAA 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 50 TAACAAG 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 51 AACAAGA 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 52 ACAAGAG 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 53 CAAGAGA 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 54 AAGAGAT 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 55 AGAGATG 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 56 GAGATGT 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 57 AGATGTG 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 58 GATGTGG 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 59 ATGTGGC 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 60 TACGTCA 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 61 ACGTCAC 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 62 CGTCACG 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 63 GTCACGT 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 64 TCACGTC 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 65 CACGTCC 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 66 AAGAGAG 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 67 AGAGAGG 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 68 GAGAGGA 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 69 AGAGGAC 7 7 bases nucleic acidNot Applicable linear Genomic DNA yes 70 GAGGACT 7 74 bases nucleic acidNot Applicable linear Genomic DNA yes 71 CTAAGTGTGG CGGAACTACGTCCTCTAACA AGAGATGTGG CGAACTACGT CACGTCCTCT 60 AACAAGAGAG GACT 74 18bases nucleic acid Not Applicable linear Genomic DNA yes 72 ACGTCCTCTAACAAGAGA 18 17 bases nucleic acid Not Applicable linear Genomic DNA yes73 TGTGGCGGAA CTACGTC 17 14 bases nucleic acid Not Applicable linearGenomic DNA yes 74 CAAGAGATGT GGCG 14 14 bases nucleic acid NotApplicable linear Genomic DNA yes 75 CTACGTCACG TCCT 14 12 bases nucleicacid Not Applicable linear Genomic DNA yes 76 CAAGAGAGGA CT 12 12 basesnucleic acid Not Applicable linear Genomic DNA yes 77 CTAAGTGTGG CG 12 9bases nucleic acid Not Applicable linear Genomic DNA yes 78 CTACGTCCT 975 bases nucleic acid Not Applicable linear Genomic DNA yes 79CTAAGTGTGG CGGAACTACG TCCTCTAACA AGAGATGTGG CGGAACTACG TCACGTCCTC 60TAACAAGAGA GGACT 75 75 bases nucleic acid Not Applicable linear GenomicDNA yes 80 CTAAGTGTGG CGGAACTACG TCACGTCCTC TAACAAGAGA TGTGGCGGAACTACGTCCTC 60 TAACAAGAGA GGACT 75 22 bases nucleic acid Not Applicablelinear Genomic DNA yes 81 TGCCAGAAGA GCCAAGGACA GG 22 20 bases nucleicacid Not Applicable linear Genomic DNA yes 82 TAAGGGTGGG CCCCTAGACC 2020 bases nucleic acid Not Applicable linear Genomic DNA yes 83CATTTGCTTC TGACACAACT 20 19 bases nucleic acid Not Applicable linearGenomic DNA yes 84 TCTCCTTAAA CCTGTCTTG 19 10 bases nucleic acid NotApplicable linear Genomic DNA yes 85 CCCTGGGCAG 10 14 bases nucleic acidNot Applicable linear Genomic DNA yes 86 GTCTTGTAAC CTTG 14 10 basesnucleic acid Not Applicable linear Genomic DNA yes 87 TGATGACTGG 10 10bases nucleic acid Not Applicable linear Genomic DNA yes 88 ATACCAACCT10

The embodiment of the invention inwhich an exclusive property ofprivilege is claimed is defined as follows:
 1. A method for detectingdisease associated alleles in patient genetic material comprising: a)immobilizing a first group of oligonucleotide molecules having a knownlength on a predetermined position on a substrate, said oligonucleotidemolecules synthesized to complement base sequences of the diseaseassociated alleles; b) contacting the patient genetic material with saidfirst group of oligonucleotides to form duplexes; c) contacting theduplexes with a second group of oligonucleotide molecules, said secondgroup of oligonucleotide molecules to noncovalently to extend the lengthof the oligonucleotide molecules of the first group, and where each ofthe oligonucleotage molecules of the second group varies from each otherin just one base position and where each of the oligonucleotidemolecules of the second group are each tagged with a fluorochrome whichradiates light at a predetermined wavelength; and d) comparing the lightpatterns radiating from the predetermined positions on the substratewith predetermined light patterns emitted from identical substrateswhich were contacted with the disease associated alleles.
 2. The methodas recited in claim 1 wherein the patient genetic material arepolynucleotides selected from the group consisting of deoxyribonucleicacid and ribonucleic acid.
 3. The method as recited in claim 1 whereinthe oligonucleotide molecules of the first group of oligonucleotides andthe oligonucleotide molecules of the second group of oligonucleotidescontain a base selected from the group consisting of guanine, cytosine,adenine, thymine, uracil, and combinations thereof.
 4. The method asrecited in claim 1 wherein the oligonucleotide molecules of the firstgroup of oligonucleotides consists of different oligonucleotidesequences and are of equal length.
 5. The method as recited in claim 1wherein the length of the oligonucleotides in the first group is between8 and 20 bases.
 6. The method as recited in claim 1 wherein theoligonucleotide molecules of the second group of oligonucleotidesconsist of different base sequences and are of equal length.
 7. Themethod as recited in claim 1 wherein the predetermined length of theoligonucleotides in the second group is between 4 and 7 bases.
 8. Themethod as recited in claim 1 wherein the fluorochrome which radiateslight at a predetermined wavelength is selected from the groupconsisting of tetramethylrodamine, fluoresceine, Texas Red, CascadeBlue, rhodamine, HEX and combinations thereof.
 9. The method as recitedin claim 6 wherein the oligonucleotide molecules of the second groupcomprised of different base sequences are tagged with the samefluorochrome.
 10. The method as recited in claim 6 wherein theoligonucleotide molecules of the second group comprised of differentbase sequences are tagged with different fluorochromes.
 11. The methodas recited in claim 1 wherein the substrate is a gel support.
 12. Themethod as recited in claim 1 wherein the oligonucleotides in the firstgroup and the oligonucleotides in the second group are further comprisedof a universal base.
 13. The method as recited in claim 12 wherein theuniversal base is selected from the group consisting of 5-nitroindole,3-nitropyrrole, inosine, or a combination thereof.
 14. The method asrecited in claim 1 wherein the oligonucleotides of the second group arecomprised of a universal base selected from the group consisting of5-nitroindole, 3-nitropyrrole, inosine or a combination thereof.